Heart disease is the leading cause of death in the United States, affecting 12 million Americans and with an annual economic burden that exceeds 110 billion dollars. Coronary heart disease accounts for the largest portion of heart disease cases. Presently, coronary artery bypass graft (“CABG”) is an accepted approach both on an elective and emergency basis for restoring blood flow to areas of the heart affected by coronary artery stenosis. CABG is one of the most common medical procedures with more than 600,000 conducted annually.
Clinically, in a CABG procedure the stenotic coronary artery is bypassed using a graft consisting of a saphenous vein or a mammary artery conduit. Unfortunately, not only is graft harvesting invasive but patients frequently lack adequate autogenous vessels to serve as bypass conduits, particularly in the case of patients who require repeated, multiple bypass procedures. Recognition of this problem led to the development of the first tissue engineering efforts almost 30 years ago. For example, Bregman and Wolinsky used subcutaneously implanted pulsed balloons to produce an autologous graft conduit via encapsulation. While their results were mixed, the concept of a tissue engineered vascular graft (“TEVG”) was established. Rather than using artificial materials, the tissue engineering approach seeks to replace pathological tissue with new tissue engineered specifically for the patient.
Twelve years later, Weinberg and Bell introduced in vitro assembly to the field by combining collagen gels with living vascular cells. Unfortunately, their grafts displayed burst strengths of less than 10 mm Hg and even the use of DACRON sleeves did not increase the burst strength of the grafts beyond 350 mm Hg. While these vessels acted as permeability barriers, the poor mechanical properties and the requirement of artificial materials limited this approach. Since the initial work by Weinberg and Bell, there have been various attempts at obtaining improved mechanical properties from completely biological, collagen gel based TEVGs. Unfortunately, the maximum demonstrated burst strength of these engineered tissues has been only 225 mm Hg, which is much lower than the 1700 mm Hg burst strength of human saphenous veins. Thus, it does not seem likely that this approach will see successful clinical application.
Other approaches have also been developed, but these approaches also have various disadvantages and insufficiencies. L'Heureux and coworkers were able to produce completely biological TEVGs by manually wrapping sheets of smooth muscle cells (“SMC”) and fibroblasts around a mandrel. Following eight weeks of culture, the mandrel was removed and endothelial cells (“EC”) were seeded on the luminal side of the graft. In vitro mechanical testing of the TEVGs showed a mean burst strength of 2594 mm Hg and histological analysis demonstrated the presence of elastin. Unfortunately, the in vivo animal study showed mixed results. The TEVGs showed “tissue-like” suturability and handling characteristics, but seven day patency was just 50%. Furthermore, problems with delamination of the layers and transport insufficiency have been observed. Finally, this approach utilized neonatal cells and the labor-intensive methodology required to use neonatal cells is impractical for large scale production of TEVGs having consistent biological and mechanical properties.
Niklason and coworkers utilized a different approach for producing TEVGs in vitro. Niklason used a tube of treated biodegradable polygalactic acid (“PGA”) polymer mesh as structural support for seeded SMCs. The tube was fitted inside a bioreactor and subjected to luminal pulsed conditions of 165 bpm, mimicking those of the fetus while in culture, for eight weeks prior to the lumen being seeded with ECs. The vessels showed some response to pharmacological agents, including serotonin, endothelin-1, and prostaglandin F2a, and demonstrated good mechanical properties including a mean burst strength above 2000 mm Hg. Histologically, the vessels exhibited collagen production, an endothelial cell layer that stained positively for von Willebrand factor and PECAM-1 (CD31), a layer of SMCs that expressed SM α-actin, heavy chain myosin, and calponin, and trace non-degraded polymer fragments. Finally, a four week in vivo study showed 100% patency for two implanted, pulsed TEVGs. While the initial results were very promising for tissues engineered with this technique, these TEVGs were not 100% biological and the degradation of the PGA mesh appeared to affect the SMCs, dedifferentiating them, perhaps due to the local hyperacid conditions. Furthermore, the culture conditions, while biomimetic, only resulted in wall shear stresses of 0.1 dyne/cm2 to 0.3 dyne/cm2, as compared to the physiological wall shear stress of approximately 10 dyne/cm2.
Campbell and coworkers used a methodology similar to that of Bregman and Wolinsky. Campbell implanted silastic tubing into the peritoneal cavity of rats and rabbits. Following harvesting after two weeks, the tubing was removed and the encapsulation layer was inverted. The resulting vessel consisted of three layers: an inner mesothelial layer that stained positively for von Willebrand factor; a middle myofibroblast layer that stained positively for α-SM actin, desmin, and heavy chain myosin; and an outer connective tissue layer, as well collagen matrix between cells. Furthermore, the TEVGs demonstrated response to pharmacological agents such as KCl, acetylcholine, and phenylephrine. The magnitude of the response to pharmacological agents was, however, much lower than that of native arteries. Overall patency rate was 67% over periods of two, three, and four months of in vivo grafting. Unfortunately, the mechanical properties of the vessels were not characterized. In addition, the cell types present in the vessel were not the same as those in native vessels. Furthermore, the assembly methodology was invasive and relied on the silastic mandrel producing a strong foreign body inflammatory response. As such, this method is not well suited for producing grafts for sick patients and is not likely to see approval by the US Food and Drug Administration for wide spread clinical use.
It is quite clear that despite their scientific value, all of the previous studies described above and known in the art have limitations regarding their ability to achieve clinical efficacy and practical application for disease management. Accordingly, there is a long felt but unmet need for improved systems and methods for in vitro generation of tissue constructs and grafts.
There is also a long felt but unmet need for methods for in vitro generation of tissue constructs and grafts that provide for tissue with high burst strengths, high patency, and other biological and mechanical properties that are consistent with in vivo use.
Additional features and objects of the invention will become apparent from the following description in which the preferred embodiments are set forth in detail in conjunction with the accompanying drawings.